This invention relates to chimeric proteins having fibrinolytic and thrombin-inhibiting properties, which are linked at the C-terminal end of the plasminogen-activating amino acid sequence to a thrombin-inhibiting amino acid sequence. The invention also relates to plasmids for producing these polypeptides and to thrombolytic agents which contain a polypeptide of this type as their active ingredient.
In all industrialized countries, cardio-circulatory diseases currently constitute the most frequent cause of death. Particularly important in this respect are acute thrombotic occlusions, the occurrence of which in the case of coronary thrombosis leads within a very short time to a life-threatening under-supply of the cardiac muscle. Similar considerations apply to cerebral thrombosis, intracerebral occlusions being accompanied here by massive ischemic damage to the brain areas concerned. In contrast to coronary thrombosis, which is associated with high mortality rates, under-supply in cerebral thrombosis does not as a rule lead to life-threatening situations, but to severe impairment of an everyday way of life due to the failure of certain brain functions, and thus leads in part to a drastic loss of quality of life for those affected. It is generally true for both these forms of thrombosis that within a few hours--without therapy--the regions supplied by the arteries concerned are irreversibly damaged. Other thrombotic occlusion diseases which require treatment include pulmonary embolism, venous thrombosis and peripheral arterial occlusion diseases.
The occlusion of a blood vessel caused by a thrombus mainly occurs at an arteriosclerotic lesion comprising fibrin, thrombocytes and erythrocytes under the action of various enzymes of the blood coagulation system. Within the enzyme cascade of the coagulation system, thrombin plays a prominent role. Thrombin can activate all the important enzymes of the coagulation system, can induce the aggregation of thrombocytes and can lead to the formation of a fibrin network by the conversion of fibrinogen to fibrin (Furie and Furie in New Engl. J. Med. 326, 800 (1992)).
The formation of thromboses is restricted by physiological anticoagulants, for example antithrombin III, activated protein C and tissue factor pathway inhibitor. Once formed, thromboses can be re-dissolved by the action of plasmin occurring naturally in the body. Plasmin is formed from an inactive proenzyme, plasminogen, which is proteolytically activated by plasminogen activators. The thrombolysis due to plasmin is utilized therapeutically, by treating patients with thrombotic diseases, particularly patients with acute coronary thrombosis, with plasminogen activators. The aim of therapeutic intervention is to reduce the infarct and to lower the mortality rate. Streptokinase, APSAC (anisolated plasminogen streptokinase activator complex), double-chain urokinase (UK), recombinant single-chain urokinase (recombinant prourokinase) and tissue plasminogen activator (t-PA) are currently available for this therapy (Collen and Lijnen in Blood 78, 3114, (1991)). It clearly follows from the experiences of lysis therapy which have been published hitherto that re-opening of the occluded coronary vessels within a few hours, i.e. 1 to 4 hours after the occurrence of the coronary, provides the best functional results. In order to achieve the aim of optimum reperfusion, therapy in the majority of cases should actually be commenced even before admission as an in-patient. However, this is only possible using a fibrinolytic agent which has few side effects and which is safe, and in view of the diagnosis situation also, which is still uncertain at this time. When employed in the requisite doses for the treatment of acute coronary disease, however, all fibrinolytic agents of the so-called first generation, such as streptokinase, APSAC and urokinase, produce a generalized plasminogen activation which is accompanied by a high risk of hemorrhage. Even the use of fibrinolytic agents of the so-called second generation, t-PA and prourokinase, leads to systemic plasminogen activation in many coronary patients. For successful reperfusion and to prevent re-occlusions, both t-PA and prourokinase have to be used in high doses, which result in significant fibrinogenolysis, and therefore to systemic plasminogen activation. This is in agreement with the observation that in previous studies no significant differences could be detected in the frequency of hemorrhage complications between patients treated with tPA or prourokinase and patients treated with streptokinase.
Various approaches have therefore been pursued aimed at improving the pharmacological profile of plasminogen activators. The following are under development: bat plasminogen activators (Gardell et al. in J. Biol. Chem. 264, 17947 (1989); Australian Patent No. AU 642,404-B (=EP 383,417), staphylokinase (Schlott et al. in Bio/Technology 12, 185 (1994); Collen and Van De Werf in Circulation 87, 1850 (1993)), the recombinant tissue plasminogen activator BM 06.022 (Martin et al. in J. Cardiovasc. Pharm. 18, 111 (1991)) and the t-PA variant TNK-t-PA (Keyt et al. in Proc. Natl. Acad. Sci. 91, 3670 (1994)).
Streptokinase, a protein of hemolytic Streptococci, activates human plasminogen, in that it forms a complex with plasminogen and thereby converts the plasminogen into an active conformation. This complex itself converts free plasminogen to plasmin, which then in turn cleaves the plasminogen bound to streptokinase. Staphylokinase, a protein obtained from Staphylococcus aureus, also acts similarly, but possesses a higher fibrin specificity compared with streptokinase. APSAC, a compound of streptokinase and human plasminogen which is produced in vitro, is a further development of streptokinase. Due to a chemical modification of the active center of the plasminogen, APSAC has a biological half-life which is longer than that of streptokinase.
Urokinase is a human protein which can be obtained in two forms as a proteolytically active protein from urine; high molecular weight urokinase (HUK) and low molecular weight urokinase (LUK) (Stump et al. in J. Biol. Chem. 261, 1267 (1986)). HUK and LUK are active forms of urokinase, i.e. double-chain molecules. Urokinase is formed as single-chain urokinase (prourokinase) in various tissues and can be detected in small amounts as a proenzyme in human blood (Wun et al. in J. Biol. Chem. 257, 3276 (1982)). As HUK, the activated form of prourokinase has a molecular weight of 54 kilodaltons and consists of 3 domains: the amino-terminal growth factor domain, the kringle domain and the serine protease domain (Guenzler et al. in Hoppe-Seyler's Z. Physiol. Chem. 363, 1155 (1982); Steffens et al. in Hoppe-Seyler's Z. Physiol. Chem. 363, 1043 (1982)). Although prourokinase and plasminogen are present as proenzymes, prourokinase is capable, due to its intrinsic activity, of transforming plasminogen into active plasmin. However, this plasminogen activator does not attain its full activity until the plasmin formed has itself cleaved the prourokinase between .sup.158 lysine and .sup.159 isoleucine (Lijnen et al. in J. Biol. Chem. 261, 1253 (1986)). The production of urokinase in Escherichia coli by genetic engineering was first described by Heyneker et al. (Proceedings of the IVth International Symposium on Genetics of Industrial Microorganisms 1982). Unglycosylated prourokinase (saruplase) is produced using a synthetic gene (Brigelius-Flohe' et al. in Appl. Microbiol. Biotech. 36, 640 (1992)).
t-PA is a protein with a molecular weight of 72 kilodaltons which is present in blood and in tissue. This plasminogen activator consists of 5 domains: the amino-terminal finger domain, the growth factor domain, kringle domain 1, kringle domain 2 and the serine protease domain. Like prourokinase, t-PA is converted into the active, double-chain form by a plasmin-catalyzed cleavage between kringle domain 2 and the serine protease domain, i.e. between .sup.275 Arg and .sup.276 Ile. In vitro studies and the results of experiments on animals indicate that t-PA binds to fibrin and its enzymatic activity is stimulated by fibrin (Collen and Lijnen in Blood 78, 3114 (1991)). The fibrin specificity of t-PA should prevent the formation of plasmin in the entire blood system, resulting not only in the decomposition of fibrin decomposed but also in the decomposition of fibrinogen. A systemic plasminogen activation such as this as well as the extensive decomposition of fibrinogen are undesirable, since this increases the risk of hemorrhage. It has been shown in therapeutic practice, however, that the expectations derived from pre-clinical studies as regards the fibrin specificity of t-PA are not fulfilled. Due to the short biological half-life of t-PA it is necessary to infuse high doses, which result in systemic plasminogen activation despite this fibrin specificity (Keyt et al. in Proc. Natl. Acad. Sci. 91, 3670 (1994)).
r-PA and TNK-t-PA are variants of t-PA which possess improved properties. In r-PA (BM 06.022) the first three t-PA domains, i.e. the finger domain, the growth factor domain and the first kringle domain, have been deleted, so that the shortened molecule only contains the second kringle domain and the protease domain. r-PA is produced in Escherichia coli by genetic engineering and is not glycosylated. Compared with t-PA, r-PA has a longer biological half-life and more rapidly leads to reperfusion. It has been shown in experiments on animals that r-PA applied as a bolus is just as effective as a t-PA infusion (Martin et al. in J. Cardiovasc. Pharmacol. 18, 111 (1991)).
The t-PA variant TNK-t-PA differs from natural t-PA on three counts: the replacement of .sup.103 threonine by asparagine, due to which a new glycosylation site is formed; the replacement of .sup.117 asparagine by glutamine, due to which a glycosylation site is removed, and the replacement of the sequence between .sup.296 lysine and .sup.299 arginine by four successive alanine units. The combination of these three mutations results in a polypeptide with a higher fibrin specificity and a longer biological half-life compared with natural t-PA. Moreover, TNK-t-PA is considerably less inhibited by PAI-1 than is natural t-PA (Keyt et al. in Proc. Natl. Acad. Sci. 91, 3670 (1994)). Results obtained from experiments on animals in which a precursor of TNK-t-PA was used indicate that TNK-t-PA is suitable for bolus application (Refino et al. in Thromb. Haemost. 70, 313 (1993)).
Bat plasminogen activator (bat-PA) occurs in the saliva of the Desmodus rotundus bat. This plasminogen activator, which has meanwhile also been synthesized by genetic engineering, has an even more pronounced fibrin specificity than t-PA and in tests on animals has exhibited improved thrombolysis with an increased biological half-life and reduced systemic plasminogen activation (Gardell et al. in Circulation 84, 244 (1991)).
In the treatment of thrombotic diseases, plasminogen activators are generally administered together with an anticoagulant substance, for example heparin. This results in improved thrombolysis compared to treatment with only a plasminogen activator (Tebbe et al. in Z. Kardiol. 80, Suppl. 3, 32 (1991)). Various clinical results indicate that, in parallel with the dissolution of thromboses, an increased tendency towards coagulation occurs (Szczeklik et al. in Arterioscl. Thromb. 12, 548 (1992); Goto et al. in Angiology 45, 273 (1994)). It is assumed that thrombin molecules which are enclosed in the thrombus and which are released again when the clot dissolves are responsible for this. Moreover, there are indications that plasminogen activators themselves also accelerate the activation of prothrombin and thus act in opposition to thrombolysis (Brommer and Meijer in Thromb. Haemostas. 70, 995 (1993)). Anticoagulant substances such as heparin, hirugen, hirudin, argatroban, protein C and recombinant tick anticoagulant peptide (TAP) can suppress this increased tendency towards re-occlusion during thrombolysis and can thus enhance the success of lysis therapy (Yao et al. in Am. J. Physiol. 262 (Heart Circ. Physiol. 31) H 347-H 379 (1992); Schneider in Thromb. Res. 64, 667 (1991); Gruber et al. in Circulation 84, 2454 (1991); Martin et al. in J. Am. Coll. Cardiol. 22, 914 (1993); Vlasuk et al. in Circulation 84, Suppl. II-467 (1991).
One of the strongest thrombin inhibitors is hirudin from the Hirudo medicinales leech, which consists of 65 amino acids. There are various iso-forms of hirudin, which differ as regards some of their amino acids. All iso-forms of hirudin block the binding of thrombin to a substrate, for example fibrinogen, and also block the active center of thrombin (Rydel et al. in Science 249, 277 (1990); Bode and Huber in Molecular Aspects of Inflammation, Springer, Berlin, Heidelberg, 103-115 (1991); Stone and Hofsteenge in Prot. Engineering 2, 295 (1991); Dodt et al. in Biol. Chem. Hoppe-Seyler 366, 379 (1985). In addition, smaller molecules derived from hirudin are known, which also act as thrombin inhibitors (Maraganore et al. in Biochemistry 29, 7095 (1990); Krstenansky et al. in J. Med. Chem. 30, 1688 (1987); Yue et al. in Prot. Engineering 5, 77 (1992)).
The use of hirudin in combination with a plasminogen activator for the treatment of thrombotic diseases is described in U.S. Pat. No. 4,944,943 (=EP 328,957) and U.S. Pat. No. 5,126,134 (=EP 365,468). The use of hirudin derivatives in combination with a thrombolytic agent is known from PCT International Patent Application WO 91/01142.
Hirullin is a protein containing 61 amino acids which is isolated from the Hirudo manillensis leech. Hirullin is identical to hirudin as regards its action and inhibitor strength, but differs very considerably from hirudin as regards its amino acid sequence. It has also proved possible to derive smaller molecules from hirullin, which are very good thrombin inhibitors (Krstenansky et al. in Febs Lett. 269, 465 (1990)).
In addition, thrombin can also be inhibited by a peptide which is derived from the amino-terminal sequence of the human thrombin receptor (Vu et al. in Nature 253, 674 (1991)). The thrombin receptor contains a thrombin-binding sequence, with an adjacent cleavage site for thrombin, in the extracellular, amino-terminal region. This sequence can inhibit thrombin provided that the cleavage site is masked by the replacement of .sup.42 serine by .sup.42 phenylalanine.
Phaneuf et al., in Thromb. Haemost. 71, 481 (1994), describe a complex which results from a fortuitous chemical linking of streptokinase and hirudin. The plasminogen-activating capacity of this streptokinase-hirudin complex is less than that of unmodified streptokinase by a factor of 8, however.
As noted above, plasminogen-activating amino acid sequences contain various domain sites which are well known and are described in the literature.
Urokinase and prourokinase comprise the following domains:
______________________________________ Domain Amino Acids Included ______________________________________ Growth Factor Domain amino acids 1 to 43 Kringle Domain amino acids 50 to 131 Serine Protease Domain amino acids 158 to 411 ______________________________________
See Guenzler et al., "The Primary Structure of High Molecular Mass Urokinase form Human Urine; The Complete Amino Acid Sequence of the A Chain", Hoppe-Seyler's Z. Physiol. Chem., 363, 1:55-65 (1982); Steffens et al., "The Complete Amino Acid Sequence of Low Molecular Mass Urokinase from Human Urine", Hoppe-Seyler's Z. Physiol. Chem., 363, 1043-1058 (1982).
Tissue plasminogen activator comprises the following domains:
______________________________________ Domain Amino Acids Included ______________________________________ Finger Domain amino acids 4 to 50 Growth Factor Domain amino acids 50 to 87 Kringle 1 Domain amino acids 87 to 176 Kringle 2 Domain amino acids 176 to 262 Serine Protease Domain amino acids 276 to 527 ______________________________________
See Collen et al., "Thrombolytic and Pharmacokinetic Properties of Human Tissue-Type Plasminogen Activator Variants Obtained by Deletion and/or Duplication of Structural/Functional Domains, in a Hamster Pulmonary Embolism Model", Thrombosis and Haeomostasis, 65, (2), 174-180 (1991).
Bat-plasminogen activator comprises the following domains:
______________________________________ Domain Amino Acids Included ______________________________________ Finger Domain amino acids 1 to 43 Growth Factor Domain amino acids 44 to 84 Kringle Domain amino acids 92 to 173 Serine Protease Domain amino acids 189 to 441 ______________________________________
See Gardell et al., "Isolation, Characterization, and cDNA Cloning of a Vampire Eat Salivary Plasminogen Activator", Journal of Biological Chem-st-y, 264, (30), 17947-952 (1989).